This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Mechanisms Leading to Enhanced Tolerance to Oxidative Stress and Increased Lifespan in Arabidopsis The metabolism of aerobic organisms leads to various risks for oxidative damage, due to the formation of reactive oxygen species (ROS). Although there is considerable evidence implicating oxidative stress in aging, there is a clear gap in our knowledge of the underlying biochemical mechanisms involved in this process. Vitamin C (ascorbate, AsA), a powerful non-enzymatic ROS scavenger, is a major contributor to the antioxidant cell capacity. Across kingdoms AsA is found at very high concentrations in multiple subcellular compartments including the mitochondria, cytoplasm, endoplasmic reticulum (ER), and chloroplast. A major objective of my group is to understand AsA metabolism and its role in conferring plants tolerance to oxidative stress and thus in delaying aging. In particular we focus on the study of the inositol pathway to AsA. Our preliminary data indicate that some of the isoforms of glucuronolactonase (GNL) and gulono-1,4-lactone oxidase (GLOase) are targeted to the mitochondria, ER, and chloroplast. The long-term goal of this project is to take advantage of the unique and powerful tools we have developed to investigate the role of subcellular AsA pools in the underlying biochemical mechanisms leading to tolerance to oxidative stress and delayed aging. We hypothesize that plants have evolved isoforms of GNL and GLOase that maintain AsA subcellular pools needed to protect essential and vulnerable molecules against oxidative stress, and that this protection is critical for the increased lifespan, enhanced growth, and extended reproductive activity displayed by the high-AsA Arabidopsis lines we have developed. Specific Aims 1[unreadable]3 will test this hypothesis. Aim 1: Investigate the role of the putative mitochondrial GLOase At5g56470 in contributing to the mitochondrial AsA subcellular pool, to protection against oxidative stress, and to increased lifespan. Aim 2: Explore the role of ER-targeted GNLs and GLOases in maintaining a redox balance conducive to proper ER function, protection against oxidative stress, and increased lifespan. Aim 3: Evaluate the role of the putative chloroplastic GNL At1g56500 in contributing to the chloroplast AsA subcellular pool, to protection against oxidative stress, and to increased lifespan. Public Health Relevance: The results of the proposed research will have direct impact on our understanding of the mechanisms by which AsA delays aging, with clear implications for human and animal health. This project will also contribute to the growing pipeline of young scientists already involved in this research, making a clear contribution to the INBRE mission.